Pharmaceutical dosage form for treatment of multiple sclerosis

ABSTRACT

The invention is directed to a method of treating a T cell-mediated autoimmune disease in animals, including humans, by the oral administration of autoantigens, fragments of autoantigens, or analogs structurally related to those autoantigens, which are specific for the particular autoimmune disease. The method of the invention includes both prophylactic and therapeutic measures.

This is a continuation, division, of application Ser. No. 08/105,912,filed Aug. 10, 1993 which is a division of application Ser. No.07/596,936, filed Oct. 15, 1990, (now abandoned); which is a CIP ofapplication Ser. No. 07/460,852, filed Feb. 21, 1990, (nowabandoned),(the national stage of PCT/US88/02139, filed Jun. 24, 1988);which is in turn a CIP of 07/065,734, filed Jun. 24, 1987, (nowabandoned).

FIELD OF THE INVENTION

The present invention relates to the field of treatment of autoimmunediseases and in particular T cell-mediated or T cell-dependentautoimmune diseases. Specifically, the present invention provides theadministration of autoantigens, or fragments or analogs thereof, for theprophylactic and therapeutic treatment of such autoimmune diseases.

BRIEF DESCRIPTION OF THE BACKGROUND ART

I. Autoimmune Diseases in General

Autoimmune diseases are caused by an abnormal immune response involvingeither cells or antibodies directed against normal tissues. A number ofstrategies have been developed to suppress autoimmune diseases, mostnotably drugs which nonspecifically suppress the immune response. Amethod of inducing immunologic tolerance by the oral administration ofan antigen to prevent autoimmune responses was first demonstrated byWells in 1911. Wells, H., J. Infect. Dis. 9:147 (1911). The oralinduction of unresponsiveness has also been demonstrated for severalT-cell dependent antigens. Ngan, J. et al., J. Immunol. 120:861 (1978),Gautam, S. et al., J. Immunol. 135:2975 (1985), Titus, R. et al., Int.Arch. Allergy Appl. Immun. 65:323 (1981). Antigen-driven peripheralimmune tolerance by the oral route has recently been shown to serve asan effective immunoregulatory therapeutic approach in severalexperimental autoimmune diseases (Higgins, P. J., et al., J. Immunol.140:440 (1988); Lider, O., et al., J. Immunol. 142:748-752 (1989);Bitar, D. M., et al., Cell. Immunol. 112:364 (1988); Nussenblatt, R. B.,et al., J. Immunol. 144:1689 (1990); Nagler-Anderson, C., et al., Proc.Nat. Acad. Sci. USA 83:7443-7446 (1986); Thompson, H. S. G., et al.,Clin. Exp. Immunol. 64:581-586 (1986)).

II. Experimental Allergic Encephalomyelitis

Scientists have also studied ways to suppress autoimmune diseases invarious animal models. Experimental allergic encephalomyelitis (EAE) isa T cell-mediated autoimmune disease directed against myelin basicprotein (MBP) and has been studied as a model for multiple sclerosis inseveral mammalian species. See, Alvord, E. et al., Experimental AllergicEncephalomyelitis--A Useful Model For Multiple Sclerosis (Allan R. Liss,New York, 1984). Immunoregulation of EAE is known to be at leastpartially dependent on suppressor T cells (Ts). It has been shown thatTs are present in rats which have recovered from EAE. Swierkosz, J. etal., J. Immuno. 119:1501 (1977). Furthermore, it has been shown thatsuppressor T cells account for the unresponsiveness to EAE that isexhibited by some mouse strains. Lando, Z. et al., Nature 287:551(1980).

Various methods have been employed to induce antigen-specificsuppression of EAE. For example, immunization with MBP emulsified inincomplete Freund's adjuvant, as shown by Lando, Z. et al., J. Immunol.126:1526 (1981), and intravenous injection of MBP-conjugated lymphoidcells as shown by Sriram, S. et al., Cell. Immunol. 75:378 (1983) havebeen used.

Three papers by Alvord et al. are reported in Annals of Neurology inVol. 6 at pp. 461-468, 468-473, and 474-482, respectively (1979). Thefirst and second of these papers disclose the suppression of EAE inmonkeys by the parenteral administration of MBP only when administeredtogether with a nonspecific adjunctive factor, e.g., an antibiotic or asteroid. The third report discloses the presence in the cerebrospinalfluid of patients with multiple sclerosis of several proteases thatdegrade MBP to antigenically active peptide fragments.

Papers by Traugott et al., J. Neurological Science 56:65-73 (1982), andRaine et al., Lab. Investigation 48:275-84 (1983) disclose thattreatment of a strain of guinea pigs suffering from chronic relapsingEAE by parenterally administered MBP alone or in incomplete Freund'sadjuvant (IFA) or in combination with a lipid hapten of myelin, namely,galactocerebroside, suppressed the clinical symptoms of EAE.

Furthermore, McKenna et al., Cell. Immun. 81:391-402 (1983), disclosesthat preinjection of rats with guinea pig MBP coupled to syngeneicspleen leukocytes or to syngeneic red blood cells suppressed thesubsequent induction of EAE using guinea pig MBP in Freund's completeadjuvant. The degree of suppression correlated positively with theamount of MBP administered.

A report by Strejan et al., Cell. Immun. 84:171-184 (1984), disclosesthat preinjection of rats with guinea pig MBP encapsulated withinphosphatidylserine liposomes suppressed the clinical signs and symptomsof EAE that appear in rats injected with guinea pig MBP in completeFreund's adjuvant.

Another paper by McKenna et al., Cell. Immun. 88:251-259 (1984),discloses that the suppressive effects of injected guinea pig MBPleukocyte complexes disclosed in their 1983 report was abolished whenanimals were pretreated with cyclophosphamide, a drug that inhibits theproduction of suppressor T lymphocytes.

A report by Krasner et al., Neurology 36:92-94 (1986) discloses thatsynthetic C copolymer I, which is being tested as a treatment formultiple sclerosis because it protects animals against EAE, does notexhibit immunologic cross-reactivity with MBP.

Additionally, Belik et al., Vopr. Med. Khim. 24:372-377 (1978),discloses the parenteral administration of "alkaline myelin proteinfragment" and "synthetic encephalitogenic peptide" to guinea pigs withEAE. The animals recovered after administration of "alkaline myelinprotein fragment" to the animals sensitized by bovine "alkaline myelinprotein fragment" or by "synthetic encephalitogenic peptide."

Previous studies in EAE and EAU demonstrated that increasing dosages ofMBP or S-Ag were associated with better disease protection (Higgins, P.J., et al., J. Immunol. 140:440 (1988); Nussenblatt, R. B., et al., J.Immunol. 144:1689 (1990)) and, in general, investigators have reportedenhancement of oral tolerance by feeding larger amounts of antigen(Mowat, A. M., Immunol. Today 8:93 (1987)).

One report has suggested that EAE may be suppressed by adoptive transferof CD8⁺ T cells from orally tolerized animals (Lider, O., et al., J.Immunol. 142:748-752 (1989)).

However, it is not known in the art to successfully treat EAE after EAEmanifests itself in the afflicted animal. Also, it is not known in theart to successfully treat multiple sclerosis after multiple sclerosismanifests itself in the patient. Thus a need still exists for a methodof suppressing and treating multiple sclerosis.

III. Adjuvant Arthritis

Adjuvant arthritis (AA) is an experimental model of inflammatory jointdisease and especially a model of rheumatoid arthritis. Adjuvantarthritis is induced by intradermal injection of a suspension ofMycobacterium tuberculosis (MT) in oil (Pearson, C. M., J. Chronic Dis.16:863-874 (1963)). Between 10 and 15 days following injection, animalsdevelop a severe, progressive arthritis.

Because of its resemblance to human rheumatoid arthritis in bothclinical and histopathological features (Jasin, H. E., Federation Proc.32:147 (1972)), AA has been used as a model to investigate mechanisms ofimmune mediated joint disease and to investigate methods for thetreatment of an organ specific autoimmune disease.

Adjuvant arthritis is a cell mediated autoimmune disease and can betransferred by cell populations or by T cell clones specific for MT(Taurog, J. D. et al., Cell. Immunol. 75:271 (1983); Taurog, J. D. etal., Cell. Immunol. 80:198 (1983); Cohen, L. R. et al., Arthritis andRhem. 28:841 (1985)). Studies have suggested that the primaryautoantigen in adjuvant arthritis is a 65-kd mycobacterial heat shockprotein (HSP) (van Eden, W. et al., Nature 331:171 (1988)). This proteinalso appears to be important in streptococcal cell wall arthritis(DeJoy, S. Q. et al., J. Exp. Med. 170:369 (1989); van den Broek, M. etal., J. Exp. Med. 170:449 (1989)). Immunity to type II collagen has beenshown to exist in adjuvant arthritis (Trentham, D. E. et al., J. Clin.Invest. 66:1109 (1980)).

Tolerization following oral and intravenous administration of collagenhas been shown to suppress another type of arthritis termedcollagen-induced arthritis (CIA). Suppression of CIA in DBA mice byorally administered type II collagens (CII) is dose-dependent withsuppression observed when 0.5 mg but not 3 mg was given 8 times over atwo-week period (Nagler-Anderson, C., et al., Proc. Natl. Acad. Sci. USA83:7443-7446 (1986)). Similar results were reported for CIA in rats withgreater protection when CII was given at 2.5 μg/g than 25 μg/g(Thompson, H. S. G., et al., Chin. Exp. Immunol. 64:581-586 (1986)). Interms of i.v. tolerization, 1 mg was given to suppress CIA in DBA mice(Myers, L. K., et al., J. Exp. Med. 170:1999 (1989)).

Adoptive transfer of protection for CIA arthritis has been reported foranimals treated intravenously with CII (Myers, L. K., et al., J.Immunol. 143:3976 (1989)) but not for oral tolerization(Nagler-Anderson, C., et al., Proc. Natl. Acad. Sci. USA 83:7443-7446(1986); Thompson, H. S. G., et al., Clin. Exp. Immunol. 64:581-586(1986)).

However, it has not previously been known that oral administration ofCII suppresses AA, the animal model for human rheumatoid arthritis, andthat this suppression can be adoptively transferred by splenic T cellsfrom CII fed animals.

Thus a need exists for the treatment of autoimmune diseases, andespecially for the treatment of T cell-mediated or T cell-dependentautoimmune disease.

SUMMARY OF THE INVENTION

The present invention provides methods for the treatment of a Tcell-mediated or T cell-dependent autoimmune disease in a subject inneed of such treatment, comprising the oral administration ofautoantigens, fragments of autoantigens, or analogs structurally relatedto autoantigens specific for the particular autoimmune disease, to suchsubject, in an amount effective to treat the autoimmune disease.

Both the clinical and histological effects of such autoimmune diseasesare suppressed in a dose-dependent manner by the methods of theinvention. Moreover, such suppression occurs whether the administrationof autoantigens occurs before or after onset of the autoimmune disease.

According to the methods of the invention, T cell-dependent autoimmunediseases are also suppressed by oral administration of nondisease-inducing and disease-inducing fragments of the autoantigen. Theoral administration of autoantigens, therefore, represents an effective,simple method by which an autoimmune disease can be naturallyimmunoregulated.

In an additional aspect of the invention, methods for the treatment andsuppression of EAE and multiple sclerosis are provided, such methodsproviding the enteral administration of specific fragments of myelinbasic protein to a subject in need of such treatment, such methods beinguseful before or after onset of the autoimmune disease.

In an additional aspect of the invention, methods for the treatment andsuppression of adjuvant arthritis and rheumatoid arthritis are provided,such methods providing the enteral administration of type II collagen(CII) to a subject in need of such treatment, such methods being usefulbefore or after onset of the autoimmune disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: antigen specificity of orally-induced suppression of theproliferative response in Lewis rats. Animals were fed 500 μg of MBP orBSA on days-7, -5 and -2, then immunized with 100 μg MBP in CFA on day0. Nine days after immunization, lymph nodes were removed andproliferative response to MBP, BSA and PPD (all at 50 μg/ml) determinedas described in Example 3. Stimulation index=experimental cpm/controlcpm.

FIG. 2: orally induced suppression of adjuvant arthritis, as measured byjoint swelling.

FIG. 3: protocol for inducing relapsing murine EAE.

FIG. 4: orally-induced suppression of lymphoid cell proliferation in SJLmice. Animals were fed 400 μg MBP 7 times over a 2 week period andimmunized with 400 μg MBP in CFA (0.6 mg/ml M. tuberculosis).Stimulation index is MBP-induced proliferation divided by background.

FIG. 5: antigen specific suppression of popliteal draining lymph nodecells (PLNC) responses by spleen and mesenteric lymph node cells (LNC)obtained from myelin basic protein (MBP) fed rats. The results areexpressed as percent suppression of PLNC to MBP (circles) as toMycobacterium tuberculosis (squares). Closed circles or closed squaresrepresent the response of spleen cells. Open circles or open squaresrepresent the response of mesenteric lymph node cells.

FIG. 6A and FIG. 6B: specific suppression of IgG responses to MBP afteroral MBP feeding. Rats were bled at intervals and sera examined foranti-OVA (FIG. 6A, open circles) or anti-MBP (FIG. 6B, open squares)antibodies. These sera were compared to sera obtained from unfed andchallenged animals (closed symbols). Results are expressed as ELISA O.D. 492 levels ± S.D.

FIG. 7: Lewis rats were fed with MT (A) or CII (B), on day-7, -5 and -2.Animals were then intradermally injected with CFA containing 10 mg/ml ofMT at the base of the tail on day 0 for induction of AA. Beginning onday 13, animals were examined for clinical signs of AA and were scoredindividually, the "arthritis score" reflects the average arthritis score(sum of the four paws) from 5-10 individual rats in each group for eachtime point.

FIG. 8: Lewis rats were fed with either buffer alone (control), orvarying dosages of CII as indicated, on days-7, -5 and -2. Animals werethen immunized intradermally with CFA containing 10 mg/ml of MT at thebase of the tail. One month later, animals were challenged with either20 μg CII (A), or 10 μg MT (B). Ear thickness was measured prior to and48 hours after injection. P values comparison of fed animals vs.control. ns=not significant.

FIG. 9: Lewis rats were fed various dosages of MT on day-7, -5 and -2and immunized on day 0 with 0.1 ml CFA at the base of the tail. Draininglymph nodes were collected 9 days later and proliferative responsesmeasured.

FIG. 10: Lewis rats were induced for arthritis by intradermal injectionof CFA containing 10 mg/ml MT. Initial signs of arthritis appeared 13-14days after disease induction. On day 17, animals were separated into twogroups with matching severity of the disease. The control group remaineduntreated whereas the treated group received 3 μg CII orally three timesper week at every other day intervals. The animals in both groups werescored for arthritis until day 34. Data are expressed as mean arthritisscore ± standard error.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

In the description that follows, a number of terms used in immunologyare extensively utilized. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

Autoimmune disease. An autoimmune disease is a malfunction of the immunesystem of an animal, including humans, in which the immune system failsto distinguish between foreign substances within the animal andsubstances which are part of the animal's normal composition.

Autoantigen. An "autoantigen" is any substance normally found within ananimal that, in an abnormal situation such as an autoimmune disease, isno longer recognized as part of the animal itself by the lymphocytes orantibodies of that animal, and is therefore attacked by theimmunoregulatory system as though it were a foreign substance.

Biologically active fragments. The term "biologically activefragment(s)" of an autoantigen includes any partial amino acid sequenceof an autoantigen that is capable of inducing the same biologicalresponse as the full-length autoantigen, i.e., the ability to suppressor eliminate T cell-mediated or T cell-dependent autoimmune response,upon oral introduction.

Analog. The term "analog(s)" of an autoantigen includes compounds thatare so structurally related to the autoantigen that they possess thesame biological activity as the autoantigen, i.e., the ability toeliminate or suppress the same or equivalent T cell-mediated or Tcell-dependent autoimmune response, upon administration of theautoantigen. As such, the term includes amino acid sequences whichdiffer from the amino acid sequence of the autoantigen by one or moreamino acids (while still retaining substantially equivalent biologicalactivity of the autoantigen) as well as chemical compounds which mimicthe biological activity of the autoantigens in their ability to suppressor alleviate the symptoms of the disease. Such compounds may consist oftissue from a target organ that is the site of attack in an autoimmunedisease.

Animal. The term "animal" covers all life forms that have animmunoregulatory system and are therefore susceptible to autoimmunediseases, including humans.

Treatment. The term "treatment" is meant to include both theprophylactic measures to prevent such autoimmune diseases as well as thesuppression or alleviation of symptoms after the onset of suchautoimmune diseases.

Administration. By the term "introduction" or "administration" of anautoantigen to a subject in need of treatment with such autoantigen isintended providing the autoantigen or its biologically active fragments,or biologically active analogs, to such subject in a manner whichretains the therapeutic effectiveness of such autoantigen for a lengthof time sufficient to provide a desired beneficial effect to suchsubject. In a preferred embodiment, the autoantigen is introduced intothe stomach of such subject by way of the mouth. However, by "oral,"Applicants do not intend to limit administration to that provided per osand intend to include any administration which provides suchautoantigens to the subject's stomach or digestive tract.

Type II collagen. Type II collagen ("CII") is the type of collagen foundinter asia, in cartilage, the interverbebral disc and the vitreous body.Type II collagen contains three α1(II) chains ( α1(II)!₃).

As is known in the art, collagen is a family of fibrous proteins thathave been classified into a number of structurally and geneticallydistinct types (Stryer, L. Biochemistry, 2nd Edition, W. H. Freeman &Co., 1981, pp. 184-199). Type I collagen is the most prevalent form andis found inter alia, in skin, tendons, cornea and bones and consists oftwo subunits of α1(I) collagen and one subunit of a different sequencetermed α2. Other types of collagen, including type II collagen, havethree identical subunits or chains, each consisting of about 1,000 aminoacids. Type III collagen is found inter alia, in blood vessels, thecardiovascular system and fetal skin and contains three α1(III) chains (α1(III)!₃). Type IV collagen is localized, inter alia, in basementmembranes and contains three α1(IV) chains ( α1(IV)!₃).

The present invention relates to the treatment of T cell-mediated or Tcell-dependent autoimmune diseases by the oral administration ofautoantigens specific for such autoimmune diseases as well asbiologically active fragments of the autoantigens, and analogs thereof.

The primary use of the invention is to treat a large category ofdiseases, prior to and/or after onset thereof, such diseases beingcollectively called autoimmune diseases, including but not limited tomultiple sclerosis, myasthenia gravis, rheumatoid arthritis, diabetesmellitus and especially juvenile diabetes mellitus, systemic lupuserythematosus, autoimmune thyroiditis, autoimmune hemolytic anemia, andcontact sensitivity disease, which may, for example, be caused by plantmatter, such as poison ivy.

Thus, according to the methods of the invention, the autoimmune responsewhich underlies multiple sclerosis may be treated by administration ofMBP or biologically active portions thereof. Also, according to themethods of the invention, the autoimmune response which underliesrheumatoid arthritis may be treated by the administration of CIIbiological active portions thereof.

The present invention is based on the discovery and confirmation thatthe oral or enteral administration of MBP is an effective means ofsuppressing chronic and acute monophasic EAE. In a highly preferredembodiment, such administration is per os. The suppression of EAE by theenteral administration of MBP after manifestation of the disease isunexpected.

The present invention is further based on the discovery that the enteraladministration of type II collagen is an effective way of suppressingadjuvant arthritis. The suppression of adjuvant arthritis by type IIcollagen is especially surprising because type II collagen isunexpectedly much more efficient at suppressing adjuvant arthritis thanis MT. In a preferred embodiment, such administration is per os.

Enterally induced tolerance in both EAE and adjuvant arthritis isdose-dependent and both clinical and histological symptoms of thedisease are lessened in severity. Because, for example, the oraladministration of an irrelevant antigen such as bovine serum albumin(BSA) or another autoantigen such as collagen or "S" antigen (theautoantigen involved with experimental autoimmune uveitis) has no effecton susceptibility to EAE, it can be said that the oral induced toleranceto EAE is specific for MBP, the antigen to which the T cells thatmediate the disease are sensitized.

Furthermore, the oral administration of MBP to rats induces thesuppression of immune responses to MBP. For example, lymphoid cellproliferation and the production of anti-MBP antibodies are bothdecreased. The cells responsible for both the suppression of the diseaseand suppression of antigen-specific cellular responses in vitro are of Tcell origin and are suppressor/cytotoxic CD8+ T lymphocytes.

Thus, as demonstrated below, using the EAE animal model for multiplesclerosis and using the animal model for AA, the simple method ofadministration of autoantigens such as MBP or CII respectively, astaught by the invention, is an effective treatment to suppress thedevelopment of specific autoimmune disease, certain immune responses tothe autoantigens, and the progression of the disease after such diseasehas manifested itself in a subject.

In general, the autoantigen, fragment, or analog is introduced orally inan amount of from one to 1000 mg per day, and may be administered insingle dose form or multiple dose form. Preferably the autoantigen,fragment, or analog is administered in an amount of from 25 to 850 mgper day. As is understood by one skilled in the art, the exact dosage isa function of the autoantigen, the age, sex, and physical condition ofthe patient, as well as other concurrent treatments being administered.Such preparations may be administered to an animal in need of treatmentfor such autoimmune disease so as to ameliorate, relieve, alleviate,reverse, or lessen the severity of the disease. Such preparations mayalso be administered to an animal who is predisposed to developing suchautoimmune disease so as to prevent the onset of such disease or tolessen the severity of such disease when it does emerge.

Where the autoantigen, fragment, or analog is introduced orally, it maybe mixed with other food forms and consumed in solid, semi-solid,suspension, or emulsion form. Such autoantigen may be mixed withpharmaceutically acceptable salts, carriers, flavor enhancers, and thelike.

An autoantigen may be administered in combination with any otherappropriate autoantigen for administration to a subject in need of such-autoantigen. For example, type II collagen(s) from more than one tissuesource or species may be used. The autoantigens of the invention mayalso be administered in combination with any appropriate pharmacologicalcarrier for administration to a subject in need of such autoantigen.Such autoantigens can be administered in any form that effectsprophylactic, palliative, preventative or curing conditions ofautoimmune disease in humans and animals.

The autoantigens of the invention can be employed in dosage forms suchas tablets, capsules, powder packets, or liquid solutions for oraladministration as long as the biological activity of the autoantigen isnot destroyed by such dosage form.

Preparations of the autoantigens of the invention for oraladministration include autoantigens provided as dry powders,food-stuffs, aqueous or non-aqueous solvents, suspensions or emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil, fish oil, and injectable organic esters. Aqueouscarriers include water, water-alcohol solutions, emulsions orsuspensions, including saline and buffered medical parenteral vehiclesincluding sodium chloride solution, Ringer's dextrose solution, dextroseplus sodium chloride solution, Ringer's solution containing lactose, orfixed oils.

Where the autoantigen, fragment, or analog is administered enterally, itmay be introduced in solid, semi-solid, suspension or emulsion form andmay be compounded with any of a host of pharmaceutically acceptablecarriers, including water, suspending agents, emulsifying agents.

The autoantigens of the invention may also be administered by means ofpumps, or in sustained-release form, especially, when administered as apreventative measure, so as to prevent the development of autoimmunedisease in a subject or when administered to ameliorate or delay analready established autoimmune disease.

Pharmaceutical compositions which contain the autoantigen of theinvention and which are useful in the methods of the invention aremanufactured in a manner which is in itself know. For example, theautoantigens may be provided as a pharmaceutical composition by means ofconventional mixing, granulating, dragee-making, dissolving,lyophilizing or similar processes. Such compositions, in and ofthemselves, find utility in the control of autoimmune disease be itchronic or acute.

Additionally, a low potency version of such compositions is useful inthe management of mild, chronic, or acute autoimmune disorders.

Autoantigens which are substantially free of natural contaminants can beisolated and purified from their natural or recombinant sources inaccordance with conventional conditions and techniques known in the artpreviously used to isolate such proteins, such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,or the like.

One of skill in the art can identify the antigenic domain(s) of anautoantigen using techniques known in the art, without undueexperimentation, and such domains are preferred in the methods of theinvention. For example, derivatives of the native autoantigens or,derivatives of recombinantly produced autoantigens can be made byproteolytic cleavage of a full-length protein with common proteases,such as, for example, trypsin, chymotrypsin, and subtilisin. Affinitychromatography with actin-derivatized resins may be used to assay suchfragments for their autoimmune disease suppression ability.

When identification of compounds or fragments thereof which possessautoimmune disease suppression activity is desired, such compounds orfragments can also be identified using techniques known in the art.

Further, such fragments may be identified by their homology to otherknown autoantigenic domains wherein it may be predicted that functionwill follow homology.

For example, autoantigens useful in the methods of the invention may beidentified by ability of such autoantigens to suppress suchautoantigen-induced autoimmune disease upon administration of suchautoantigen to a subject afflicted with or predisposed to the autoimmunedisease. In the methods of the invention, autoimmune disease may besuppressed by such administration of autoantigen either prior to orafter appearance of disease symptoms.

Having now generally described the invention, the following examplesfurther describe the materials and methods used in carrying out theinvention. The examples are not intended to limit the invention in anymanner.

EXAMPLES

A. Methodology

Animals: Female Lewis or Wistar Furth rats weighing 150 to 220 g (6-8weeks of age) were obtained from Charles River Laboratory, Wilmington,Mass., or from Harlan Sprague Dawley, Inc., Indianapolis, Ind., and usedin all experiments.

Immunization of Animals: Rats were immunized in both hind footpads with50 μg guinea pig MBP emulsified in complete Freund's adjuvant (CFA). Insome experiments, 50 μg ovalbumin (OVA) (Sigma) was added to theemulsified antigens and injected similarly. EAE was characterized bylimb paralysis and scored as follows: 0) no disease; 1) decreasedactivity, limp tail; 2) mild paralysis, unsteady gait; 3) moderateparaparesis, limbs splayed apart; and 4) tetraplegia.

Induction of Oral Tolerance: Rats were fed MBP or bovine serum albumin(BSA) five times at three-day intervals 1 mg in 1 ml PBS (8 gm NaCl 0.2gm KCl, 1.44 gm of Na₂ HPO₄, 0.24 gm of KH₂ PO₄ in 1000 ml of H₂ O)using a 23-gauge needle covered with plastic tubing.

Proliferation Assay: Nine days after immunization, the rats weresacrificed and their popliteal lymph nodes were removed. A single cellsuspension was prepared by pressing the lymph nodes through a stainlesssteel mesh. A total of 10⁵ lymph node cells (LNC) were cultured with theindicated number of either irradiated (2000 Rads) or intact LNC derivedfrom fed rats in quadruplicate in round-bottomed 96-well plate (Costar).MBP and Mycobacterium tuberculosis (Mt), 50 μg/ml were added to theculture in a volume of 20 μl. The cultures were incubated for 80 hoursand were pulsed with 1 μCi ³ H! TdR/well for the last 16 hours ofculture. The cultures were then harvested on an automatic cell harvesterand read on a standard liquid scintillation counter.

Percent suppression of primed LNC (PLNC) proliferation was calculated bythe following formula: ##EQU1##

Proliferation Media: RPMI (Gibco) was used in all the experiments. Themedium was filtered sterile after adding 2×10⁻⁵ M 2-mercaptoethanol, 1%sodium pyruvate, 1% penicillin and streptomycin, 1% non-essential aminoacids, and 1% autologous serum.

Purification of Different Cell Subsets: For depletion of CD3, CD4, andCD8 populations from spleen cells, negative selection was used. Petridishes were coated overnight at 40° C. with 10 ml of 1/1000 goatanti-mouse IgG+IgM antibodies (Tago) in PBS/BSA. The plates were thenwashed and coated with 3% fetal bovine serum in PBS for 30 min at 20° C.and washed again. Lewis LNC were stained with mouse anti-rat monoclonalantibodies (Serotec/Bioproducts) for CD3 (MRC, OX/38), CD4 (W 3/25), orCD8 (OX/8) diluted 1/100 in PBS. The cells were stained for 30 min onice, washed, and seeded on the precoated petri dishes, 15 millioncells/5 ml PBS/plate, at 4° C. The supernatant containing nonadherentcells was aspirated gently 60 minutes later and centrifuged twice beforecell examination and counting. This protocol yields cell populations ofabout 85-95% purity as examined in the fluorescence activated cellsorter by examining membrane immunofluorescence.

Adoptive Transfer Experiments: Donor rats were fed with either MBP orBSA, 1 mg×5 times, at 3-4 day intervals and sacrificed 4 days after thefinal feeding. Mesenteric LNC and spleen cells were harvested andinjected intraperitoneally either immediately or after activation withconcavalin-A (Con-A), 1.5 μg/ml, in proliferation media for 48 hrs. Thenumber of cells injected for adoptive transfer experiments were asfollows: 120×10⁶ for whole LNC population, either activated or not;60×10⁶ for CD3 depleted LNC; 80×10⁶ for CD4 depleted population; and95×10⁶ for CD8 depleted LNC. Recipient Lewis rats were immunized withBP/CFA 4 hrs later for the induction of EAE.

Serum Levels of Antibodies: A solid-phase enzyme-linked immunoabsorbentassay (ELISA) was used for determination of antibody titers against MBPand OVA. Microtiter plates were incubated with 0.1 ml per well of 10 μgantigen/ml in doubled distilled water. Plates were incubated for 18 hrsat 25° C. After 3 washes with PBS/tween-20 (Bio-Rad), pH 7.5, plateswere incubated with 3% BSA/PBS for 2 hrs at 37° C., washed twice, and100 μl of diluted serum was added in quadruplicate. The plates wereincubated for 2 hrs at 37° C. After three rinses with PBS/tween-20,plates were incubated with 100 μl/well of peroxidase-conjugated goatanti-rat IgG antibody (Tago, USA) diluted 1:1000 in 1% BSA/PBS for 1 hrat 25° C. Color reaction was obtained by exposure to D-phenylenediamine(0.4 mg/ml phosphate) citrate buffer, pH 5.0) containing 30% H₂ O₂. Thereaction was stopped by adding 0.4N H₂ SO₄ and OD 492 nm was read on anELISA reader.

In Vitro Measurement of Antibody Production: Popliteal and splenic LNCwere obtained from fed, naive and challenged rats and seeded at aconcentration of 10⁷ cells per ml petri dish either alone or irradiated(2000 Rads) together with other PLNC as indicated. The cultures weremaintained in proliferation media, with or without antigen (20 μg/ml),for 3 days in an incubator and then harvested. The diluted supernatantswere used to examine the in vitro production and secretion of IgGantibody and were measured for antibody production using an ELISA testas described previously.

Identification of Different Regions of the Myelin Basic Protein MoleculeResponsible for Suppression of EAE: Overlapping fragments of the 1-37region of guinea pig myelin base protein were synthesized using solidphase peptide technique. Houghten, R., Proc. Natl. Acad. Sci. USA82:5131-5135 (1985). These fragments were then administered orally inequimolar concentrations to 15 mg of whole myelin basic protein. Theywere administered on day-7, -5, and -2 prior to immunization. Animalswere then challenged with basic protein in Freund's adjuvant accordingto established procedures and scored.

Demonstration that Oral Route of Administration of a Protein AntigenDetermines to Which Fragment There Is an Immune Response: Animals weregiven whole myelin basic protein, either immunized in the foot pad withFreund's adjuvant or administered orally. Seven to 10 days thereafter,spleen and lymph node cells were removed and restimulated in vitro withdifferent fragments of the myelin basic protein molecule.

Collagens and Adjuvant: Soluble-form chicken type II collagen wasobtained from Genzyme Corporation, Boston, Mass. Bovine type IIIcollagen was purchased from Southern Biotechnology Associates, Inc.,Birmingham, AL, whereas the type I collagen was a gift from Dr. D.Trentham, Beth Israel Hospital, Boston, Mass. Mycobacterium tuberculosisand incomplete Freund's adjuvant (IFA) were purchased from DifcoLaboratories, Detroit, Mich. Complete Freund's adjuvant (CFA) wasprepared by mixing IFA and MT ground to a fine powder.

Oral Administration Protocol: Antigens were orally administered in a 1ml volume through a syringe fitted with 18G ball-point needle threetimes (on days-7, -5 and -2) before induction of disease. Collagens weredissolved in potassium phosphate buffer (pH 7.6) whereas MT wassuspended in phosphate-buffered saline (PBS) for the feedings.

Induction of Arthritis: Adjuvant arthritis was induced in animals byintradermal injection at the base of the tail with 0.1 ml CFA containing10 mg/ml M. tuberculosis.

Evaluation of Arthritis. The incidence of arthritis was defined as thenumber of rats that had clinical evidence of arthritis within 35 daysafter induction of disease. The severity of arthritis was gradedaccording to standard methodology (Trentham, D. E. et al., J. Exp. Med.146:857 (1977)). Each of the four paws was graded as follows: 0=normal,1=redness only, 2=redness plus mild swelling, 3=severe swelling, 4=jointdeformity. The arthritis score for each animal was the sum of the scorefor each of the four paws. The maximum arthritis score was the highestscore of an individual animal during the entire course of the disease.All evaluations were performed in a blinded fashion without knowledge ofthe treatment group.

Lymphocyte Proliferation Assay: Rats were primed with 0.1 ml CFAcontaining 1 mg/ml MT at the base of the tail. Nine days later, thedraining lymph nodes were removed and single cell suspensions wereprepared. After being washed twice, the cells were resuspended in RPMI1640 containing 1% glutamine, 1% penicillin/streptomycin, 1%non-essential amino acids, 5% fetal calf serum and 5×10⁻⁵ M2-mercaptoethanol. The cells were then seeded into a 96-well flat-bottomplate in quadruplicate at the concentration of 2.5×10⁵ cells/well andcultured with various concentrations of MT at 37° C. with 5% CO₂ for 72hours. Tritiated thymidine was then added to the culture at 1 μCi/well.The cells were harvested 6 hours after the pulsing and proliferation wasdetermined by tritiated thymidine incorporation as measured by liquidscintillation counting.

Delayed-type Hypersensitivity (DTH) Responses: DTH responses weremeasured 30 days after the immunization. Rats were injectedsubcutaneously in both ears with either 10 μg of MT of 20 μg CII in 50μl PBS. Ear swelling consisted of the difference in ear thicknessmeasured before and 48 hours after the injection using micrometercaliper. DTH responses were also performed in unimmunized animals andanimals fed CII only.

Adoptive Transfer of Suppression: Donor rats were fed three times with 3μg of CII at 2-3 day intervals. Their spleens were removed 7 days afterthe last feeding and a single cell suspension was prepared. After lysisof the red blood cells with tris-NH₄ Cl, ph 7.26 the splenocytes werewashed twice in Hank's balanced salt solution (HBSS). In someexperiments the splenocytes were further separated into T or B cellenriched populations by using nylon wool columns. 1×10⁸ cells wereinjected intraperitoneally to each recipient, which were then injectedwith CFA to induce arthritis either on the same day or 2 days later.Splenocytes from unfed normal rats served as a control.

Example 1 Effect of Feeding MBP and Fragments Thereof

The effect of feeding MBP and its peptic fragments on the susceptibilityto and severity of acute monophasic EAE was studied in the Lewis rat.Results show that this natural route of tolerance induction suppressesboth the development of disease and immune responses to MBP.

To orally induce suppression of EAE, Lewis rats were fed MBP purifiedfrom guinea pig brain (Diebler, G., et al., Prep. Biochem. 2:139 (1972))using a syringe equipped with a 20G ball point needle. Control animalswere fed equal amounts of bovine serum albumin (BSA) or saline alone.EAE was induced by immunization with 50 μg MBP emulsified in completeFreund's adjuvant (CFA) containing 200 μg Mycobacterium tuberculosis byinjection into the hind footpads. Disease was characterized by hind limbparalysis and incontinence usually between days 12 and 15 afterimmunization and in all cases rats recovered by day 16. The first seriesof experiments investigated the effect of number of feedings and dose ofMBP on disease expression. Rats were fed various amounts of MBP eitheronce 7 days before (day-7) the day of immunization (day 0) or threetimes on days-14, -7 and 0. The results (Table I) demonstrate thatfeeding MBP to rats suppresses EAE and that orally-induced suppressionis dose-dependent. Multiple 500 μg feedings resulted in completesuppression of disease and were more effective than a single feeding atthis dose. In addition to clinical manifestation of EAE, histologicalevidence of disease in rats was examined. Sixteen days afterimmunization, rats were sacrificed and brains removed and fixed informalin solution. Fixative was a solution of 100 ml 70% ethanol, 10 ml37% formalin and 5 ml glacial acetic acid. Slides of paraffin-embeddedtissue were prepared from each rat and stained with hematoxylin andeosin. Perivascular inflammatory foci were quantified on coded slides byestablished procedures (Sobel, R., et al., J. Immunol. 132:2393 (1984)).As shown in Table I, feeding rats 500 μg MBP on days-14, -7 and 0 causeda marked decrease in the number of inflammatory lesions in the brain. Amoderate decrease was found in animals fed 100 μg and no significantreduction of inflammation was found in rats fed 25 μg MBP.

Example 2 Effect of Prior Exposure to Antigen on Suppression

A second series of experiments investigated the effect of feeding MBPprior to or subsequent to immunization with MBP to determine whether theeffectiveness of orally-induced suppression is affected by priorexposure to antigen. For these experiments, animals were fed 500 μg MBPthree times either before or after active induction of disease(immunization with MBP). The results (Table II) demonstrate that theclinical expression of disease is suppressed whether animals were fedMBP before or after sensitization, the effect being more complete whenantigen was fed prior to immunization. However, histologic examinationrevealed a dramatic reduction of perivascular infiltrates in rats fedMBP either before or after sensitization to MBP. Greater than 60%suppression of disease also occurred when rats were fed three timesbeginning on days +5 or +7 after immunization (data not shown).

In addition, experiments were performed in which rats were fed 100 μg ofMBP at various times, before and after immunization, with MBP. As shownin Table III, disease suppression is seen with single feedings before orafter immunization.

Example 3 Effect of Oral Administration of MBP on Cellular and HormonalImmune Responses to MBP

The effects of oral administration of MBP on cellular and humoral immuneresponses to MBP were also examined. Proliferative responses to MBP werestudied after feeding rats different doses of MBP and following feedingat different times with respect to immunization. Ten days afterimmunization, rats were sacrificed and single cell suspensions ofdraining (popliteal) lymph nodes prepared. Cells were cultured inmicrowells for 4 days, the final 24 hours with ³ H-thymidine added. Avolume of 0.2 ml containing 4×10⁵ cells in RPMI 1640 containing 2%glutamine, 1% penicillin/streptomycin, 5×10⁻⁵ M 2-mercapto-ethanol and5% fetal calf serum was added to each microwell and MBP added at 50ug/ml. Wells were pulsed with 1 μCi tritiated thymidine, harvested ontofiberglass filters using a multiharvester and counted using standardliquid scintillation techniques.

Results (Tables I and II) demonstrate that feeding MBP causes apronounced (75-92%) decrease in proliferative responses to MBP.Suppression of proliferation, unlike suppression of disease, occurred atall doses and feeding regimens tested, including feeding afterimmunization. Orally-induced suppression of the proliferative responseto MBP is antigenspecific, as shown in FIG. 1. Specifically, feeding MBPdoes not suppress the proliferative response to purified proteinderivative (PPD), an antigen derived from M. tuberculosis that induces aproliferative response as a consequence of immunization with CFA.Feeding an irrelevant antigen, BSA, does not affect the proliferativeresponse to PPD and only slightly suppresses the proliferative responseto MBP.

Example 4 Effect of Feeding MBP on the Production of Antibody to MBP

The effect of feeding MBP on the production of antibody to MBP was alsoexamined. Rats fed MBP were immunized and blood removed by cardiacpuncture 16 days following immunization. Levels of anti-MBP antibody inthe serum were measured by ELISA. A volume of 0.1 ml of MBP solution(0.05 mg/ml in PBS) was added per microwell and incubated for 3 h at 37°C. Wells were washed with PBS containing 0.05% Tween (PBST) and blockedovernight at 4° C. with 5% BSA in PBS, pH 9.0. After washing wells withPBST, diluted rat sera were added and incubated for 3 h at r.t. andafter washing with PBST secondary antibody (peroxidase conjugated goatanti-rat) added for 1 h at r.t. Substrate was added and the reaction wasstopped with 0.1M NaFl. Plates were read at 450 nm on a Titertekmultiscan. Abs₄₅₀ was also determined for serum from rats immunized onlywith CFA and was subtracted from all values as background.

Unlike suppression of proliferative responses which occurred atvirtually all doses and feeding regimens tested, suppression of antibodyproduction was only observed when animals were fed the highest dosetested (500 μg) on days-14, -7, and 0 (66% suppression, Table I). Ofnote is the lack of suppression in rats fed 500 μg MBP on days-7, -5 and-2 (Table II) suggesting that the temporal sequence in which anidentical dose of MBP is fed is important in suppression of antibodyresponses.

                  TABLE I    ______________________________________    Effect of Feeding Dose on Orally-Induced    Suppression of EAE in Lewis Rats                           Immune Response                           to MBP             Induction of EAE                           (percent inhibition)             .sup.a Clinical                     .sup.b Histologic                               .sup.c Proli-                                        .sup.d Anti-             Disease Score     feration body    ______________________________________    Immunized Controls               19/22     9.2 ± 5.8                                   --     --    Fed day -7     25 μg  3/5       ND        75.6 ± 2                                          ND    100 μg  2/5.sup.e *                         ND        88.9   ND    500 μg    3/10*** ND        88.9 ± 2                                          ND    Fed days -14, -7, 0     25 μg  3/5       7.2 ± 5.2                                   82.1   -48 ± 72    100 μg   2/5*     3.2 ± 1.9                                   80.8 ± 5                                          14 ± 49    500 μg    0/10*** 0.2 ± 0.4                                   87.2 ± 1                                          66 ± 39    ______________________________________     .sup.(a) Rats were fed various doses of MBP on the indicated days and     immunized with 50 μg MBP in CFA (200 μg M. tuberculosis on day 0.     Shown are the number of diseased rats of the total number immunized.     Immunized controls were fed BSA or saline.     .sup.(b) Rats were sacrificed on day 16 after immunization and brains     removed and fixed. Shown are the average number of perivascular     inflammatory foci per animal +/- s.d. ND = not determined.     .sup.(c) Proliferative response to MBP was measured for draining lymph     node cells ten days after rats were immunized. A volume of 0.2 ml     containing 4 × 10.sup.5 cells in RPMI 1640 containing 2% glutamine,     1% penicillin/streptomycin, 5 × 10.sup.-5 M 2mercapto-ethanol and 5     fetal calf serum was added to each microwell and MBP added at 50 μg/ml     Wells were pulsed with 1 μCi tritiated thymidine, harvested onto     fiberglass filters using a multiharvester and counted  using standard     liquid scintillation techniques. Shown is the percentage inhibition of     proliferative response to MBP with respect to the immunized control group     Average stimulation index of the immunized controls (MBPstimulated     cpm/background cpm) was 6.0 (29,888 cpm/4960 cpm).     .sup.(d) Rats were sacrificed on day 16 and blood drawn by cardiac     puncture. Sera were diluted 1/15,625 in PBS and antiMBP antibody levels     were determined by ELISA. A volume of 0.1 ml of MBP solution (0.05 mg/ml     in PBS) was added per microwell and incubated for 3 h at 37° C.     Wells were washed with PBS containing 0.05% Tween (PBST) and blocked     overnight at 4° C. with 5% BSA in PBS, pH 9.0. After washing wells     with PBST, diluted rat sera were added and incubated for 3 h at  room     temperature and after washing with PBST secondary antibody (peroxidase     conjugated goat antirat) added for 1 h at room temperature. Substrate was     added and the reaction was stopped with 0.1M NaFl. Plates were read at 45     nm on a Titertek multiscan. Abs.sub.450 was also determined for serum fro     rats immunized only with CFA and was subtracted from all values as     background. Shown is the percentage decrease in antibody level, as     measured by absorbance of peroxidase substrate at 450  nm, with respect t     immunized controls (Mean absorption at A.sub.450 of immunized controls     with background subtracted was 0.148).     .sup.(e) Groups were compared by chisquare analysis with one degree of     freedom: *p < .05, **p < 0.1, ***p < .001.

                  TABLE II    ______________________________________    Effect of Feeding MBP to Rats Before or After    Immunization on the Development of EAE                            Immune Response                            to MBP              Induction of EAE                            (percent inhibition)              .sup.a Clinical                      .sup.b Histologic                                .sup.c Proli-                                         .sup.d Anti-              Disease Score     feration body    ______________________________________    Immunized Controls                23/26     21.6 ± 5.1                                    --     --    Days fed 500 μg MBP    -7, -5, -2, +2, +5, +7                0/5.sup.e ***                          0.2 ± 0.4                                    ND     34    -7, -5, -2    0/17*** 0         92.6   15    +2, +5, +7    4/10**  1.4 ± 2.3                                    91.5 ± 3                                           15    ______________________________________     .sup.(a) Rats were fed 500 μg MBP on the indicated days and immunized     with 50 μg MBP in CFA on day 0. Immunized controls were fed BSA or     saline.     .sup.(b) See Table I.     .sup.(c) See Table I. Average stimulation index of immunized controls was     9.4 (82,247 cpm/8,718 cpm).     .sup.(d) See Table I. Mean absorption at A.sub.450 of immunized controls     with background subtracted was 0.403.     .sup.(e) See Table I.

                  TABLE III    ______________________________________    Orally Induced Suppression of EAE in Lewis Rats    Feeding Schedule                   # Rats Sick/Total    ______________________________________    None           11/16    -14, -7, 0, +7  0/13    -14            1/5    -7             0/5    0              1/5    +7             1/5    ______________________________________     Rats were fed 100 μg MPB on the indicated days (with respect to day of     immunization = 0), and immunized with 50 μg MBP with CFA (.5 mg/ml M.     tuberculosis).

Example 5 Persistence of Orally-Induced Protection Against EAE

Further experiments were conducted to determine the persistence oforally-induced protection against EAE. After feeding on days-7, -5 and-2 with 500 μg MBP rats were immunized at various lengths of time afterthe last feeding. EAE was completely suppressed in rats for up to fourweeks after feeding, and by eight weeks 50% of rats fed MBP were againsusceptible to disease. The results are shown in Table IV, whichindicates that tolerance to the disease is maintained for at least fourweeks after the last feeding, with susceptibility to disease inductionbecoming apparent at eight weeks following feeding.

                  TABLE IV    ______________________________________    Persistence of Orally Induced Tolerance of Lewis Rats                  # Rats Sick/Total    ______________________________________    Control          9/14    Fed    Immunized day 0 0/4    day +7          0/4    day +14         0/4    day +28         0/3    day +56         4/8    ______________________________________     Rats were fed 500 μg MBP on days -7, -5, and -2 and immunized on the     indicated days with 50 μg MBP in CFA. Control rats (fed BSA) were     likewise immunized.

Example 6 Effect of Fragments of MBP on the Development of EAE

It is known that the encephalitogenic region of guinea pig MBP in ratsis a specific decapeptide sequence located at residues 75-84, which byitself can induce EAE, whereas other regions of the molecule arenon-encephalitogenic (Hashim, G., Myelin: Chemistry and Biology, Alan R.Liss, N.Y. (1980)). Furthermore, for other antigens, it has beenreported that distinct suppressor determinants exist at sites differentfrom immunogenic determinants (Yowell, R., et al., Nature 279:70(1979)). It was therefore investigated whether both encephalitogenic andnon-encephalitogenic fragments of MBP could prevent EAE via oraladministration. Fragments of guinea pig MBP were generated by limitedpepsin digestion and separated by column chromatography (Whitaker, J.,et al., J. Biol. Chem. 250:9106: (1975)). The three different fragmentswere fed to rats, then animals were immunized with whole MBP. It wasfound that both the disease-inducing (fragment 44-89) andnon-encephalitogenic (fragments 1-37 and 90-170) peptides suppressed EAEwhen fed to rats, the non-encephalitogenic fragments being moreeffective in suppressing the disease than the encephalitogenic fragment(Table V). A decapeptide (S79) was synthesized which differs from theencephalitogenic sequence (residues 75-84) by a single amino acidsubstitution and is reported to induce suppression when injected intorats with CFA (Kardys, E., et al., J. Immunol. 127:862 (1981)). When S79(Ala-Gln-Gly-His-Arg-Pro-Gln-Asp-Glu-Gly) was fed to animals it was alsofound to suppress EAE (Table V). Bovine MBP, which differs from guineapig MBP at several sites including the encephalitogenic sequence and isnot encephalitogenic in rats at doses encephalitogenic for guinea pigMBP (Holoshitz, J., et al., J. Immunol. 131:2810 (1983)), alsosuppressed disease when fed to animals prior to immunization.

                  TABLE V    ______________________________________    The Effect of Feeding Encephalitogenic and    Non-Encephalitogenic Fragments on the Development    of EAE in Lewis Rats                    Clinical Incidence of EAE    ______________________________________    Immunized Controls                      19/25    MBP fragment 1-37 (109 μg)                      0/9.sup.a ***    MBP fragment 44-89 (135 μg)                        3/11**    MBP fragment 90-170 (235 μg)                       0/4**    Peptide S79 (30 μg)                        1/8***    Bovine MBP (500 μg)                        0/10***    ______________________________________     Lewis rats were fed the indicated amounts of MBP fragments or peptides     (equimolar to 500 μg whole guinea pig MBP) on days -7, -5 and -2 and     immunized on day 0 with 50 μg guinea pig MBP with CFA. Shown are the     number of diseased rats of the total number immunized.     .sup.(a) Groups were compared to immunized controls by chisquare analysis     **p < .01, ***p < .001.

Example 7 Suppression of Adjuvant Induced Arthritis by FeedingMycobacteria

Adjuvant arthritis was induced in female Lewis rats by immunization with0.1 ml of 10 mg/ml of complete Freund's adjuvant in the base of thetail. Animals were fed 2.0 mg of Mycobacteria tuberculosis in phosphatebuffered saline on days-7, -5, and -2 prior to immunization on day 0 andsubsequent to immunization on days+7 and +14. Arthritis was quantitatedby measuring joint swelling for three weeks following immunization(Table VI and FIG. 2). Subsequent studies have indicated that while theresults shown in FIG. 2 are occassionally obtained, in most instances,adjuvant arthritis was not suppressed by feeding animals Mycobacteriatuberculosis. Therefore, the ability to suppress adjuvant arthritis withMycobacteria tuberculosis administration is highly variable. The reasonfor this variability is unknown.

                  TABLE VI    ______________________________________                 Joint swelling (mm) on day 21    ______________________________________    Control        7.61 ± 1.4    Days Fed Mycobacteria    -7, -5, -2     5.61 ± 1.1*    -7, -5, -2, +7, +14                   6.07 ± 0.9*    ______________________________________     Joint swelling = thickness of joint on day measured     *p < 0.01 compared to control (representative experiment of 4     animals/group)

Example 8 An Adoptive Transfer Model of EAE in the SJL Mouse

A workable, reproducible model of adoptive relapsing EAE was establishedin the SJL mouse. The protocol for this model was adopted fromMokhtarian, et al., Nature 309:356 (1984). This protocol is depictedgraphically in FIG. 3. Briefly, donor animals are immunized with anemulsion containing 400 μg of MBP and 30 μg of M. tuberculosis in CFA.Ten days thereafter, draining lymph nodes are removed and cultured with50 ug/ml of MBP for four days, washed extensively, and 4-6×10⁷ viablecells are injected intravenously into female recipient animals. Animalsare scored for clinical EAE using standard scales, and scoredpathologically using standard H & E histological analysis (Brown, A., etal., Lab Invest. 45:278 (1981), Lublin, F., et al., J. Immunol. 126:819(1981), and Bernard, C. et al., Eur. J. Immunol. 16:655 (1976)). Animalsare monitored for at least 100 days after transfer so that the number ofrelapses can be determined.

Example 9 Orally Induced Suppression of Proliferative Responses in SLJMice

The feeding of 400 μg MBP every other day for two weeks (total of sevenseparate feedings) prior to immunization with 400 μg MBP in CFA (0.6mg/ml M. tuberculosis) suppresses the proliferation of lymph node cellsin response to MBP immunization. The results are shown in FIG. 4. ThisFigure depicts the control results versus the feeding results as afunction of the MBP-induced proliferation divided by background(Stimulation Index).

The invention is not limited to those modes and embodiments of thisapplication and embodiments that have been described above. Itencompasses any modifications that result in the suppression ofautoimmune diseases as taught by the present invention. Theseequivalents are included within the field of protection defined by theclaims.

Example 10 Adoptive Transfer of Protective Resistance to EAE Developmentfrom MBP Fed Donor Rats to Naive Syngeneic Recipient Rats

Donor rats were fed with either MBP or BSA, 1 mg×5 times, at 3-4 dayintervals and sacrificed 4 days after the final feeding. Mesentericlymph node cells (LNC) and spleen cells were harvested and injectedintraperitoneally either immediately or after activation withconcanavalin-A (Con-A), 1.5 μg/ml, in proliferation media for 48 hrs.The number of cells injected for adoptive transfer experiments were asfollows: 120×10⁶ for whole LNC population, either activated or not;60×10⁶ for CD3 depleted LNC; 80×10⁶ for CD4 depleted population; and95×10⁶ for CD8 depleted LNC. Recipient Lewis rats were immunized withMBP/CFA 4 hrs later for the induction of EAE. The ability to transferresistance to development of EAE from fed donor rats to naive syngeneicrecipient rats is shown in Table VII. LNC obtained from unfed rats orfrom bovine serum albumin (BSA) fed donor rats failed to transferprotection against EAE. However, both spleen cells or mesenteric (MES)lymph node cells obtained from MBP fed donors were capable oftransferring relative protection against EAE induced in the recipients,demonstrating 50% and 57% suppression of disease, respectively. The meanmaximal severity of disease was also reduced markedly in recipients ofeither spleen cells or mesenteric lymph nodes cells obtained from MBPfed donor rats. These results demonstrate that the oral tolerance to EAEinduction is of cellular origin and that the cells responsible forprotection are found to be concentrated in both the mesenteric lymphnodes and the spleen.

                  TABLE VII    ______________________________________    Adoptive transfer of protection against EAE using LNC obtained    from either fed or untreated donor rats.    Rats    Donors      EAE in Recipients    Fed with            Source of LNC                        Incidence                                 Mean Max. severity    ______________________________________    None    SPC         6/7      2.5 ± 0.3            Mes.LNC     5/5      2.6 ± 0.4    BSA     SPC         4/4      2.4 ± 0.2            Mes.LNC     5/5      2.6 ± 0.3    MBP     SPC          4/8*     1.6 ± 0.2*            Mes.LNC      4/7*     1.7 ± 0.2*    ______________________________________     Lewis rats were fed with either MBP or BSA five times, 1 mg per feeding a     3 day intervals, or remained untreated. The rats were then sacrificed and     their spleens and mesenteric lymph nodes were removed. The LNC were     harvested and activated for 48 hours in the presence of ConA. The     lymphoblasts were collected, washed three times, and injected     intraperitoneally into naive syngeneic rats. The recipient rats were     challenged 4 hours later with MBP/CFA for the induction of EAE. The     disease  was scored daily from day 10 (*Results are statistically     significant, p < 0.05).

Example 11 Identification of the Lymph Node Cell SubDopulation whichmediates Resistance to EAE

Con-A activated spleen cells (SPC) obtained from MBP fed donor rats weretransferred to naive syngeneic rats either before or after depletingeither T cells, helper T lymphocytes (CD4) or suppressor/cytotoxic Tlymphocytes (CD8). For depletion of CD3, CD4 and CD8 populations fromspleen cells, negative selection was used. Petri dishes were coatedovernight at 4° C. with 10 ml of 1/1000 goat anti-mouse IgG+IgMantibodies (Tago) in PBS/BSA. The plates were then washed and coatedwith 3% fetal bovine serum in PBS for 30 min at 20° C. and washed again.Lewis LNC were stained with mouse anti-rat monoclonal antibodies(Serotec/Bioproducts) for CD3 (MRC, OX/38), CD4 (W3/25) or CD8 (OX/8)diluted 1/100 in PBS. The cells were stained for 30 min on ice, washedand seeded on the precoated petri dishes, 15 million cells/5 mlPBS/plate, at 4° C. The supernatant containing nonadherent cells wasaspirated gently 60 minutes later and centrifuged twice before cellexamination and counting. This protocol yields cell populations of about85-95% purity as examined in the fluorescence activated cell sorter byexamining membrane immunofluorescence. The results are demonstrated inTable VIII. The results demonstrate that SPC are capable of transferringprotection against EAE (50% incidence), whereas T cell depleted SPC losttheir ability to protect recipient rats (group 2). Thus, it seems thatthe spleen cells which are capable of transferring protection are Tlymphocytes. However, depletion of CD8 cells (group 4) results infailure of transferring protection, whereas CD4+ depleted SPC showed asignificant ability of protecting rats against EAE. Thus, it is evidencethat the antigen specific T lymphocytes which are generated after oraladministration of MBP and which are mediating resistance to diseaseinduction are of the suppressor/ cytotoxic subset.

                  TABLE VIII    ______________________________________    Adoptive transfer of protection against EAE using depleted    population of SPC.    SPC removed from EAE in recipient rats    Group  MBP fed donors                         Incidence Mean Max. Severity    ______________________________________    1      Whole population                         2/4       1.7 ± 0.2*    2      CD3 depleted  6/6       2.6 ± 0.4*    3      CD4 depleted   2/6*     1.2 ± 0.2*    4      CD8 depleted  6/7       2.2 ± 0.3    ______________________________________     Donor rats were fed with MBP, and treated as indicated in the legend of     Table 1. The ConA activated SPC were injected into naive recipient rats     either before (group 1) or after depletion of certain subpopulation     (groups 2-4). Depletion of CD3, CD4 or CD8 lymhocytes was done by couplin     monoclonal IgG antibodies to the SPC and panning. Recipient rats were     immunized with MBP/CFA and EAE was recorded from day 10 (*Results are     statistically significant, p < 0.05).

Example 12 In vitro Suppression of Anti-MBP T Cell Responses by Additionof Lymph Node Cells from MBP Fed Rats

Rats were immunized with MBP/CFA and their primed popliteal draininglymph nodes (PLNC) harvested nine days later. A single cell suspensionwas prepared by pressing the lymph nodes through a stainless steel mesh.A total of 10⁵ LNC were cultured with the indicated number of eitherirradiated (2000 Rads) or intact LNC derived from fed rats inquadriplicate in round bottomed 96-well plate (Costar). MBP andMycobacterium tuberculosis, 50 μg/ml were added to the culture in avolume of 20 μl. The cultures were incubated for 80 hrs. and were pulsedwith 1 μCi ³ H! TdR/well for the last 16 hours of culture. The cultureswere harvested on an automatic cell harvester and read on a standardliquid scintillation counter.

Percent suppression of primed LNC (PLNC) proliferation was calculated bythe following formula: ##EQU2## The PLNC were cultured along withirradiated SPC or mesenteric LNC obtained from either naive or MBP fedrats in the presence of either MBP or Mycobacterium tuberculosis. TheLNC obtained from MBP fed donor rats were examined on a different daysafter last feeding. Results are shown in FIG. 5. It is shown that withinthe time frame of the experiment, LNC obtained from fed rats did notaffect the PLNC responses to Mycobacterium tuberculosis. However, bothSPC and mesenteric LNC obtained from fed rats were able to suppress thePLNC proliferation to MBP. Antigen specific suppression of PLNCresponses was greater using SPC than mesenteric LNC. Suppression isevident from day 5 to day 36 after the last feeding with MBP indicatingthat the induction of suppression is achieved soon after feeding and itis maintained for a relatively long period of time.

Thus, it seems that LNC obtained from rats rendered to be tolerized toEAE induction are antigen-specific lymphocytes which are capable ofsuppressing cellular immune responses only to the antigen used forfeeding.

Example 13 Suppression of Anti-MBP Responses of PLNC in the Presence ofIrradiated SPC and its Subpopulations, Obtained from a MBP Fed Rat

To examine the subpopulation of SPC responsible for suppression, SPCwere obtained from MBP fed rat 20 days after the last feeding, depletedof certain lymphocyte populations, irradiated and mixed with PLNCobtained from MBP/CFA immunized rat together with MBP. Popliteal andsplenic LNC were seeded at a concentration of 10⁷ cells per ml petridish either alone or irradiated (2000 Rads) together with other PLNC asindicated. The cultures were maintained in proliferation media, with orwithout antigen (20 μg/ml), for 3 days in an incubator and thenharvested. The diluted supernatants were used to examine the in vitroproduction and secretion of IgG antibody and were measured for antibodyproduction using an ELISA test. Microtiter plates were incubated with0.1 ml per well of 10 μg antigen/ml in doubled distilled water. Plateswere incubated for 18 hrs. at 25° C. After 3 washes with PBS/tween-20(Bio-Rad), pH 7.5, plates were incubated with 3% BSA/PBS for 2 hrs. at37° C., washed twice and a 100 μl of diluted serum was added inquadruplicate. The plates were incubated for 2 hrs. at 37° C. Afterthree rinses with PBS/tween-20, plates were incubated with 100 μl/wellof peroxidase-conjugated goat anti-rat IgG antibody (Tago, USA) diluted1:1000 in 1% BSA/PBS for 1 hr. at 25° C. Color reaction was obtained byexposure to D-phenylenediamine (0.4 mg/ml phosphate citrate buffer, pH5.0) containing 30% H₂ O₂. The reaction was stopped by adding 0.4N H₂SO₄ and the OD 492 nm was read on an ELISA reader. The results shown inTable IX represents the percent suppression of the antigen proliferationof PLNC in the presence of SPC obtained from MBP fed rats compared totheir responses to MBP in the presence of SPC obtained from intact rats.It is demonstrated that SPC obtained from MBP fed rats (group 1)suppresses the responses of PLNC to MBP (70%). Depletion of T cells(group 2) or suppressor/cytotoxic T lymphocytes (group 3) abrogatessuppression. However, depletion of helper T lymphocytes (CD4, group 4)enhances the inhibition of the anti-MBP proliferation response of thePLNC. Diluting the CD4 depleted SPC results in decreasing of suppressionfrom 96% (in the 1:1 ratio) to 18% (in the 1:100 ratio of SPC:PLNC).

These results suggest that the cells responsible for both diseaseinhibition and antigen-specific cellular responses in vitro are of the Tcell origin and that they are suppressor/cytotoxic T lymphocytes.

                  TABLE IX    ______________________________________    Suppression of anti-MBP responses of PLNC in the presence of    irradiated SPC and its subpopulations, obtained from MBP fed rats.          SPC removed from                       SPC:PLNC  % Suppression of PLNC    Group MBP fed rats ratio     responses to MBP    ______________________________________    1     Whole population                       1:1       70    2     CD3 depleted 1:1       -13    3     CD8 depleted 1:1       -30    4     CD4 depleted 1:1       96          "             1:10     32          "             1:50     35          "             1:100    18    ______________________________________     Spleens were removed from MBP fed Lewis rats, then cells were harvested,     irradiated and seeded along with responder PLNC removed from MBP/CFA     immunized syngeneic rats. The SPC were used as untreated cells or deplete     of CD3, CD4 or CD8 T lymphocytes using the appropriate monoclonal     antibodies for coupling and then panning. Results are expressed as percen     suppression of PLNC responses to MBP and are relative to the PLNC     responses in the presence of irradiated SPC removed from unfed rats .

Example 14 Humoral Suppression of Anti-MBP IgG Production Induced byOral Tolerance to MBP

Lewis rats were either fed with MBP or left untreated and thenchallenged with MBP mixed with ovalbumin (OVA) emulsified in CFA. Therats were then bled at various intervals, and sera was examined foranti-OVA or anti-MBP antibodies. As shown in FIG. 6a, the IgG serumlevels to OVA were not affected in MBP fed rats, whereas IgG serumlevels to MBP were decreased in MBP fed rats (6b).

Example 15 Determination of the Cell Type Responsible for theSuppression of IgG Production In Vitro

Lewis rats were fed with MBP or remained unfed and then were immunizedwith MBP+OVA/CFA. The PLN were removed 12 days later, and the PLNC werecultured for 3 days in the presence of either MBP or OVA, thesupernatants were collected, diluted 1:20 and examined for their IgGcontents. As shown in Table X, PLNC, which were obtained from fed rats(group 2) and cultured in vitro with MBP, responded less in terms of IgGproduction to MBP in comparison to PLNC obtained from unfed rats (group1, 45% suppression). The production of anti-OVA IgG production in PLNCfrom the same rats was not affected, (group 4 vs. 5). Moreover, mixingirradiated PLNC obtained from MBP fed and immunized rats with PLNC ofimmunized rats cultured together with MBP, decreased the antibodyproduction of the later (group 3, 35% suppression), whereas theantibodies titers against OVA was not affected (group 6). In addition,removal of CD8+ cells abrogated the suppression of anti-MBP antibodiesdemonstrating that, as in adoptive transfer and proliferative responses,CD8+ cells were responsible for suppression.

                  TABLE X    ______________________________________                    IgG Levels in Supernatants                                             %                                             Sup-                                             pression                              In Vitro       of IgG          Responder Modulator Stimu-                                    O.D. 492 Pro-    Group Cells     Cells     lation                                    Values ± S.D.                                             duction    ______________________________________    1     Immunized --        MBP   0.56 ± 0.06                                             --    2     MBP Fed   --        MBP   0.31 ± 0.01                                             45          and          Immunized    3     Immunized MBP Fed   MBP   0.36 ± 0.04                                             35                    and                    Immunized    4     Immunized MBP Fed   MBP   0.55 ± 0.04                                              0                    and                    Immunized                    CD8.sup.+                    depleted    5     Immunized --        OVA   0.17 ± 0.03                                             --    6     MBP Fed   --        OVA   0.18 ± 0.02                                              0          and          Immunized    7     Immunized MBP Fed   OVA   0.21 ± 0.04                                              0                    and                    Immunized    ______________________________________     Rats were immunized with MBP + OVA and CFA (some 3 days after the fifth     feeding of MBP). Twelve days later their PLNC were removed and cultured     together with MBP (groups 1-4) or with OVA (groups 5-7) for three days. I     some groups, irradiated PLNC obtained from MBP fed and immunized rats wer     irradiated and cultured along with immunized PLNC in the presence of MBP     (group 3) or in the presence of OVA (group 7). The supernatants of these     stimulations were collected, diluted and  IgG levels determined by ELISA.

Example 16 Identification of the MBP Region which Actively SuppressesEAE using Overlapping Synthetic Polypeptides of MBP

Overlapping fragments of the amino acid 1-37 fragment of guinea pigmyelin basic protein were synthesized using solid phase peptidetechnique. Houghten, R., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985).These fragments were then administered orally in equimolarconcentrations to 15 mg of whole myelin basic protein. They wereadministered on day-7, -5, and -2 prior to immunization. Animals werethen challenged with basic protein in Freund's adjuvant according toestablished procedures and scored.

Animals were scored for mortality, presence of disease, and diseaseseverity. As shown in Table XI, 6/6 control animals became ill with amortality of 3/6. In animals receiving overlapping peptide fragments,there was decreased mortality using all fragments, except for fragment1-10. When viewed in terms of disease severity, the region of themolecule between amino acids 5 and 20 shows the most pronounceddiminution of disease. These results demonstrate that in the amino acidregion 1-37 which itself is a suppressogenic fragment, specific regionsof the molecule may be more or less suppressive when administeredorally.

                  TABLE XI    ______________________________________                     EAE Mediated by MBP/CFA               Incidence   Mean Max.    Fragment   of Disease  Score     Mortality    ______________________________________    Control (PBS)               6/6         3.8       3/6     1-10      5/5         3.8       4/5     5-15      4/5         2.1       1/5    11-20      4/5         2.0       0/5    16-25      4/5         2.6       0/5    21-30      5/5         3.0       1/5    26-36      4/6         2.6       1/6    31-37      5/6         3.3       0/6    ______________________________________     Overlapping fragments of the 1-37 region of guinea pig myelin basic     protein were synthesized using solid phase peptide technique. These     fragments were then administered orally in equimolar concentrations to 15     mg of whole myelin basic protein. They were administered on day -7, -5,     and -2 prior to immunization. Animals were then challenged with basic     protein in Freund's adjuvant according to established procedures and     scored.

Example 17 Demonstration that Oral Route of Administration of a ProteinAntigen Determines to which Fragment there is an Immune Response

Animals were given whole myelin basic protein, either immunized in thefoot pad with Freund's adjuvant or administered orally. Seven to 10 daysthereafter, spleen and lymph node cells were removed and restimulated invitro with different fragments of the basic protein molecule.

As shown in Table XII, when myelin basic protein is administeredperipherally in Freund's adjuvant, the primary response is to the 44-89encephalitogenic region as measured by proliferation. However, as shownin Table XIII, when it is administered orally, the primary response isto fragment 1-37, the non-encephalitogenic suppressor determinant.

                  TABLE XII    ______________________________________    Proliferation to MBP fragments in Lewis rats immunized with    whole MBP.                Counts Per Minute                             Stimulation Index    ______________________________________    Background     3,292         --    Whole MBP     10,142         3.1    MBP fragment 1-37                   3,360         1.0    MBP fragment 44-89                  10,054         3.0    ______________________________________     Animals were immunized in hind foot pads with 50 μg MBP in CFA. Ten     days later lymph nodes were removed and stimulated in vitro with 10 μg     MBP or equimolar amounts of MBP fragments.

                  TABLE XIII    ______________________________________    Proliferation to MBP fragments in Lewis rats fed whole MBP orally.    Source of LNC               Whole MBP    1-37     44-89    ______________________________________    SPC        5.10 ± 1.6                            5.05 ± 1.8                                     2.41 ± 0.9    Mes.LNC    8.61 ± 1.9                            9.88 ± 1.5                                     3.53 ± 0.8    Cervicals  4.58 ± 1.3                            6.42 ± 0.9                                     2.51 ± 0.6    ______________________________________     Animals were fed 1 mg of whole MBP x3, then cells removed from various     organs 15 days following feeding and proliferation measured. Results are     expressed as the change in cpm × 10.sup.-3 as compared to cells     cultured alone.

Example 18 The Effect of Feeding Mycobacterium tuberculosis or Type IICollagen on Adiuvant Arthritis

Since AA is induced by CFA containing M. tuberculosis, an initialapproach in studying the problem was to feed animals with variousdosages of MT. Unexpectedly, no suppression of disease was observed asmeasured by incidence of arthritic limbs, day of onset, or maximumarthritis score over a wide dose range, in which 3 μg, 30 μg, 300 μg or3 mg of MT was administered on days-7, -5 and -2 prior to immunization.Representative data in which animals were pretreated with 3 μg is shownin FIG. 7A.

Based on investigations which reported the development of autoimmunityto collagen in rats with AA (Trentham, D. E. et al., J. Clin. Invest.66:1109 (1980)), the effect of orally administering type II collagen onAA was studied. As shown in FIG. 7B and Table XIV, prefeeding rats withCII significantly suppressed AA in a dose-dependent manner with the mostpronounced effects seen in groups fed 3 μg or 30 μg of CII. Occasionalsuppression was seen at 300 μg. In animals fed 3 μg or 30 μg, theincidence of arthritic limbs was less and the disease was midler asmeasured by the maximum arthritis score. The onset of the disease wasalso delayed in animals fed 3 μg of CII. To determine whether oraladministration of CII had non-specific suppressive effects onexperimental autoimmune diseases, an identical dose-range of CII was fedto animals immunized with myelin basic protein in CFA for the inductionof experimental autoimmune encephalomyelitis (EAE) (Higgins, P. J. etal., J. Immunol. 140:440 (1988)). No effect on the development of EAEwas observed following feeding CII.

                  TABLE XIV    ______________________________________    The Effect of Feeding Collagen II on Adjuvant Arthritis               arthritic            maximum    pre-treatment               limbs      day of onset                                    arthritis score    ______________________________________    control    40/40      13.1 ± 0.3                                    9.1 ±  1.2    (buffer alone)    CII 0.3 μg               19/20      12.6 ± 0.2                                    9.6 ±  1.4    CII 3 μg               .sup. 26/36.sup.a                          .sup. 15.3 ± 1.1.sup.b                                    .sup. 5.1 ±  0.9.sup.c    CII 30 μg               .sup. 30/40.sup.a                          13.7 ± 0.4                                    .sup. 6.2 ±  0.7.sup.b    CII 300 μg               39/40      13.1 ± 0.3                                    7.7 ±  0.9    CII 1 μg               19/20      12.4 ± 0.2                                    9.0 ±  1.6    ______________________________________     Lewis rats were fed with either buffer (control group), or various doses     of CII three times on days -7, -5 and -2, and intradermally injected on     day 0 at base of the tail with CFA containing 1 mg of MT for the inductio     of adjuvant arthritis. The arthritis was evaluated every 2-3 days from da     12 to day 31. pvalues represent CII fed groups vs. controls (PBS fed).     ns = not significant.     .sup.a p < 0.001 vs. control     .sup.b p < 0.05 vs. control     .sup.c p < 0.01 vs. control

Example 19 Delayed Type Hypersensitivity Responses Following OralAdministration of Type II Collagen and MT

It has been reported that immunity to both CII and MT develops in AA(Trentham, D. E. et al., J. Clin. Invest. 66:1109 (1980)). DTH responseswere performed to determine the effect of feeding CII on in vivo T cellresponses to both MT and CII. As shown in FIG. 8A, animals immunizedwith CA develop DTH to CII although it is not as pronounced as DTH to MT(FIG. 8B). Furthermore, oral administration of CII reduced the DTHresponse to CII in animals with AA, whereas there was no effect on theDTH response to MT. The dose response range for suppression of DTH byCII was identical as for suppression of disease with CII, i.e., the mostprominent suppression seen at 3 μg and 30 μg. Of note is that there wasno sensitization to CII in animals that were only fed 3 μg withoutsubsequent immunization. The suppression of cellular immune responses toMT following oral administration of the antigen was next examined. Asshown in FIG. 9, the proliferative responses to MT were suppressed inanimals fed with 3 μg and 30 μg MT. Similar suppression was observed asmeasured by DTH responses.

Example 20 Adjuvant Arthritis is Suppressed by Adoptive Transfer of Tcells from CII Orally Tolerized Rats

It was previously shown that suppression of EAE following oraladministration of myelin basic protein can be adoptively transferred bysplenic T cells from fed animals (Lider, O, et al., J. Immunol.142:748-752 (1989)), and similar results in the autoimmune uveitis modelhave been obtained. As shown in Table XV, protection against AA wasadoptively transferred to naive rats by splenic T cells from rats orallytolerized to CII. Protection was more pronounced when splenocytes weretransferred on day-2 and when splenic T cells vs. B cells weretransferred.

                  TABLE XV    ______________________________________    The Effect of Feeding Non-cartilaginous Collagens on Adjuvant Arthritis               Arthritic            Maximum    Pretreatment               Limbs      Day of Onset                                    Arthritis Scores    ______________________________________    Control    20/20      12.6 ± 0.7                                    9.7 ± 1.9    (buffer alone)    CI 3 μg .sup.  9/16.sup.a                          16.3 ± 3.6                                    .sup. 5.2 ± 2.5.sup.b    CI 30 μg               18/20      14.0 ± 1.5                                     4.2 ± 0.6.sup.c    CIII 3 μg               18/20      .sup. 14.4 ± 0.5.sup.b                                    7.2 ± 2.0    CIII 30 μg               18/20      .sup. 14.5 ± 0.6.sup.b                                    6.3 ± 0.7    ______________________________________     Lewis rats were fed with varying degrees of CI or CIII three times on day     -7, -5, and -2 (control animals were fed buffer only). pvalues represent     fed vs control.     ns = not significant.     .sup.a p < 0.01 vs. control     .sup.b p < 0.04 vs. control     .sup.c p < 0.001 vs. control

Example 21 Suppression of AA by Oral Administration of CII After DiseaseOnset

In order to determine if feeding CII could ameliorate alreadyestablished AA, animals were fed CII after the onset of disease. Initialsigns of arthritis appeared 13-14 days after disease induction with CFA.On day 17, animals were separated into two groups with matching severityof the disease. The control group remained untreated whereas the treatedgroup received 3 μg CII orally three times per week at every other dayintervals. The animals in both groups were scored for arthritis untilday 34. As shown in FIG. 10, animals treated with CII developed milderarthritis and recovered sooner than controls.

Example 22 The Effect of Feeding Non-cartilage Collagens on AA

Even though the molecular structure of CII is very closely related toother collagens, such as type I (CI) and type III collagen (CIII), thedistribution of these collagens is quite different (Seyer, J. M., etal., In: Textbook of Rheumatology, 3rd ed. (Kelly et al., eds.), p. 22,Saunders, Philadelphia (1989)). Whereas CII is usually present in thecartilage of the joints, type I and type III collagens are found mostlyin bones, skin, and other soft tissues. As shown in Table XVI, we foundthat oral administration of CI suppressed AA, as determined by incidenceof arthritic limbs and disease severity, in the same dose range as CII.There was a delay in disease onset in animals fed CIII but nosignificant effect on disease severity. Oral administration of anirrelevant protein antigen, myelin basic protein, did not suppress AA.

                  TABLE XVI    ______________________________________    Suppression of Adjuvant Arthritis by Adoptive Transfer    of Splenocytes from CII-orally Tolerized Rats                    Day of  Arthri-           Cells    Trans-  tic   Day of  Arthritis    Donor  Transferred                    fer     Limbs Onset   Score    ______________________________________    Exper-    iment I    1 Normal           Spleno-   0      20/20 13.6 ± 0.2                                          11.2 ± 1.5           cytes    2 CII fed           Spleno-   0      17/20 14.0 ± 0                                           6.6 ± 0.9.sup.a           cytes    3 Normal           Spleno-  -2      20/20 13.8 ± 0.2                                          9.2 ± 1.6           cytes    4 CII fed           Spleno-  -2      .sup.  15.2 ± 0.7.sup.c                                           2.8 ± 0.5.sup.c           cytes    Exper-    iment II    1 Normal           Spleno-  -2      20/20 13.8 ± 0.2                                          9.0 ± 2.0           cytes    2 CII fed           Splenic B                    -2      20/20 13.8 ± 0.2                                          8.8 ± 1.1           cells    3 CII fed           Splenic T                    -2      16/20 14.0 ± 0.5                                          4.0 ± 0.5.sup.d           cells    ______________________________________     Donor Lewis rats were either unfed (normal) or prefed three times at 2-3     day intervals with 3 μg of CII. Spleens were taken 7 days after the     last feeding and 1 × 10.sup.8 splenocytes or nylon wool separated B     (adherent) or T (nonadherent) cells were transferred by i.p. injection to     each recipient which were induced for AA immediately or 2 days after     adoptive transfer.     .sup.a p < 0.05, group 2 vs. group 1     .sup.b p < 0.001, group 4 vs. group 3     .sup.c p < 0.01, group 4 vs. group 3     .sup.d p < 0.05, group 3 vs. group 1

The lymphocyte proliferation and DTH experiments above indicated thatoral administration of MT suppressed cellular immune responses againstMT without inhibition of clinical disease. Nonetheless, cellularimmunity to MT was not profoundly suppressed by oral tolerance and itmay be that regimens that had a greater effect on suppressing MTimmunity would suppress disease. In this regard, others have shownsuppression of AA by administering the 65 kd HSP in oil (Billingham, M.E. J., et al., J. Exp. Med. 171:339 (1990) or by administering MTintradermally or intravenously (Larsson, P., et al., J. Cell.Biochemistry 40:49 (1989); Gery, I., et al., Int. Arch. Allergy 31:57(1967)).

Suppression of AA by oral administration of CII suggests either thatpathogenic immunity to CII develops in AA or that there arecross-reactive epitopes between MT and CII. Of note is that CII T celllines were reported to have a minor effect in ameliorating M by T cellvaccination (Holoshitz, J., et al., Science 219:56 (1983) and there wasslight suppression of CIA by the 65 kd HSP (Billingham, M. E. J., etal., J. Exp. Med. 171:339 (1990)). Some investigators have reportedsuppression of AA by intravenous administration of CII (Phadke, K., etal., Arthritis Rheum. 27:797 (1984)) although this has not beenuniformly found (Cremer, M. A., et al., J. Immunol. 131:2995 (1983)).Our studies suggest that the inability of investigators to demonstratesuppression of AA by i.v. administration of CII (Cremer, M. A., et al.,J. Immunol. 131:2995 (1983)) may relate to the use of too large a dose,viz., 1 mg. In preliminary experiments, some cross-reactivity between MTand CII has been found in proliferation assays although it remainsundefined as to whether the suppression of AA by oral administration ofCII relates to cross-reactivity between MT and CII. Amino acid sequencehomology between chicken type II collagen and peptide 180-188 of the 65kd heat shock protein of MT, which has been reported to stimulate clonesmediating arthritis in rats (van Eden, W., et al., Nature 331:171(1988)) has not been found. Recently, a 26-amino acid sequence from CIIhas been reported to suppress collagen induced arthritis (Myers, L. K.,et al., J. Exp. Med. 170:1999 (1989)), however, no homologies betweenthis peptide and the 65 kd peptide can be located. Clearly, given thesize of both MT and CII, cross-reactive epitopes may exist which are noteasily identified. Alternatively, MT may induce joint damage that leadsto a pathogenic immune response to CII.

It has been demonstrated that active suppression is generated followingoral administration of antigen (Ngan, J., et al., J. Immunol. 120:861(1978); Mattingly, J. A., et al., J. Immunol. 125:1044 (1980);Mattingly, J. A., Cell. Immunol. 86:46 (1984); Zhang, Z., et al., Cell.Immunol. 104:426 (1987)), and that EAE may be suppressed by adoptivetransfer of CD8⁺ T cells from orally tolerized animals (Lider, O, etal., J. Immunol. 142:748-752 (1989)).

Example 23 Treatment of Multiple Sclerosis Patients

The medication used for treatment is a bovine myelin extract prepared byBioPure, Boston, Mass. Bovine myelin is non-toxic when administered toanimals and is effective in ameliorating chronic relapsing EAE.BioPure's bovine myelin is prepared on a sucrose gradient via densitycentrifigation using a Sharples centrifuge and analyzed by SDS pageelectrophoresis. The myelin is extracted from bovine brains obtainedfrom local slaughter houses in Massachusetts and tested for purity andbatch to batch standardization by agarose gel electrophoresis, proteindetermination, lipid analysis, amino acid determination, and immunologicreactivity. It is also tested for the presence of bacteria and viruses.

The myelin is administered to patients with multiple sclerosis in 100 mgcapsules given three times per day for a total dose of 600 mg/day.

Example 24 Treatment of Autoimmune Arthritis Patients

The type II collagen used for treatment is a CII preparation obtainedfrom Genzyme Corporation, Boston, Mass. (soluble chicken type IIcollagen). This preparation is effective in ameliorating adjuvantarthritis.

The CII is administered orally to patients with autoimmune arthritis ina dose of 10 μg to 100 mg per day. The CII is administered in a dry formor dissolved in a liquid (and volume) the patient is able to tolerate.In a preferred embodiment, a total dose of 100 μg up to 30 mg per day isadministered. Such dosage may be administered in multiple doses so as toprovide the patient with the total daily dose. In a preferredembodiment, such multiple dosage is three times per day.

All references cited herein are fully incorporated herein by reference.Having now fully described the invention, it will be understood by thosewith skill in the art that the scope may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

What is claimed is:
 1. A method for the treatment of multiple sclerosisin a human comprising orally or enterally administering myelin to saidhuman in an amount effective to treat said disease.
 2. The method ofclaim 1 wherein said treatment suppresses the clinical symptoms of saiddisease.
 3. The method of claim 1 wherein said myelin is administeredorally.
 4. The method of claim 1 wherein said myelin is administered ina solid oral dosage form.
 5. A solid or semi-solid pharmaceutical dosageform adapted for oral administration for the treatment of multiplesclerosis in a human comprising an effective amount for treating saidmultiple sclerosis of myelin.
 6. The solid or semi-solid pharmaceuticaldosage form of claim 5 wherein said dosage form is selected from thegroup consisting of a capsule, a tablet, and a powder packet.
 7. Thesolid or semi-solid pharmaceutical dosage form of claim 5 wherein saidmyelin is a bovine myelin.
 8. The solid or semi-solid pharmaceuticaldosage form of claim 5 comprising about 100 mg of said myelin per unitdosage.
 9. The solid or semi-solid pharmaceutical dosage form of claim 5further comprising a pharmaceutically acceptable carrier.
 10. A solid orsemi-solid pharmaceutical dosage form adapted for oral administrationfor the treatment of multiple sclerosis in a human comprising aneffective amount for treating said multiple sclerosis of a compositioncomprising MBP.
 11. The pharmaceutical dosage form of claim 10, whereinsaid MBP is a purified MBP.
 12. The pharmaceutical dosage form of claim10, wherein said MBP is a substantially pure MBP.
 13. The pharmaceuticaldosage form of claim 10, wherein said MBP is recombinantly produced. 14.The pharmaceutical dosage form of claim 10 wherein said dosage form isselected from the group consisting of a capsule, a tablet, and a powderpacket.